This invention is directed towards a method and kit for determining the nucleotide base sequence of a nucleic acid, particularly for use in routine clinical diagnostic procedures.
DNA sequence-based diagnosis has the potential to become a routine clinical diagnostic test, and is already available in several formats. See, for example, U.S. Pat. Nos. 5,545,527, 5,550,020 and 5,552,283 which are incorporated herein by reference. To fully realize the potential for DNA sequence-based diagnostics, however, the development of simplified, and preferably single-tube sequencing techniques will be important.
DNA sequencing technique are generally well known. Such sequencing is generally performed using techniques based on the "chain termination" method described by Sanger et al., Proc. Nat'l Acad. Sci. (USA) 74(12): 5463-5467 (1977). Basically, in this process, DNA to be tested is isolated, rendered single stranded, and placed into four vessels. In each vessel are the necessary components to replicate the DNA strand, i.e., a template-dependant DNA polymerase, a short primer molecule complementary to a known region of the DNA to be sequenced, and individual nucleotide triphosphates in a buffer conducive to hybridization between the primer and the DNA to be sequenced and chain extension of the hybridized primer. In addition, each vessel contains a small quantity of one type of dideoxynucleotide triphosphate, e.g. dideoxyadenosine triphosphate(ddA).
In each vessel, each piece of the isolated DNA is hybridized with a primer. The primers are then extended, one base at a time to form a new nucleic acid polymer complementary to the isolated pieces of DNA. When a dideoxynucleotide is incorporated into the extending polymer, this terminates the polymer strand and prevents it from being further extended. Accordingly, in each vessel, a set of extended polymers of specific lengths are formed which are indicative of the positions of the nucleotide corresponding to the dideoxynucleic acid in that vessel. These sets of polymers are then evaluated using gel electrophoresis to determine the sequence.
Improvements to the original technique described by Sanger et al. have included improvements to the enzyme used to extend the primer chain. For example, Tabor et al. have described enzymes such as T7 DNA polymerase which have increased processivity, and increased levels of incorporation of dideoxynucleotides. (See U.S. Pat. No. 4,795,699 and EP-A1-0 655 506, which are incorporated herein by reference). More recently, Reeve et al. have described a thermostable enzyme preparation, called Thermo Sequenase.TM., with many of the properties of T7 DNA polymerase. Nature 376: 796-797 (1995). The literature supplied with the Thermo Sequenase.TM. product suggests dividing a DNA sample containing 0.5-2 mg of single stranded DNA (or 0.5 to 5 mg of double stranded DNA) into four aliquots, and combining each aliquot with the Thermo Sequenase.TM. enzyme preparation, one dideoxynucleotide termination mixture containing one ddNTP and all four dNTP's; and a dye-labeled primer which will hybridize to the DNA to be sequenced. The mixture is placed in a thermocycler and run for 20-30 cycles of annealing, extension and denaturation to produce measurable amounts of dye-labeled extension products of varying lengths which are then evaluated by gel electrophoresis.
DNA sequencing can be performed using these procedures on genomic DNA or cDNA (a DNA copy of mRNA). Alternatively, the direct sequencing of mRNA is known using techniques similar to DNA sequencing.
When low levels of substrate template are present, it is generally necessary to amplify its amount before sequencing reactions can be reliably performed. A well known method of amplifying a DNA strand is by the polymerase chain reaction ("PCR"). PCR methods are disclosed in U.S. Pat. Nos. 4,683,194, 4,683,195 and 4,683,202, which are incorporated herein by reference. RNA amplification may be effectively performed using techniques disclosed in U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517 (See also Sooknanan, R., van Gemen, B., and Malek, L. T. "Nucleic Acid Sequence Based Amplification" in Molecular Methods for Virus Detection chp. 12 (Academic Press; 1995)), also incorporated herein by reference. A related RNA amplification method is disclosed by Gen-Probe patents WO 9101384, WO 9525180, WO 9503430, U.S. Pat. No. 5,399,491, incorporated herein by reference.
The instant invention discloses a simplified method of determining the sequence of a nucleic acid in a patient sample, in a one-pot or single tube sequencing reaction that does not rely on the PCR method.
It is an object of the invention to provide a method and kit for determining the nucleic acid sequence of an RNA molecule in an RNA sample obtained from a patient sample.